1. Field of the Invention
The present application relates to synthesis of decitabine (also known as 2′-deoxy-5-azacytidine; 5-aza-2′-deoxycytidine; DAC; 5-aza-dC; dezocitidine; and 4-amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-one)), which is an active pharmaceutical ingredient (API) useful, among other things, in treating myelodysplastic syndromes (MDS).
2. Description of the Related Arts
A number of methods have been developed to synthesize decitabine but these methods, on the whole, are inefficient and less desirable for commercial production. One important problem is that when the 5-azacytosine ring (s-triazine ring) is conjugated to a carbohydrate, it is sensitive to decomposition by water (under neutral, basic and acidic conditions) and in fact undergoes facile hydrolysis in aqueous formulations, aqueous emulsions, aqueous solutions, and when exposed to moisture during aqueous work-up. This problem makes commercial manufacture of 5-azacytosine based-nucleosides challenging.[1],[2] Another problem in decitabine synthesis is that the key glycosyl donor (carbohydrate ring) and nucleobase coupling reaction that forms the nucleoside itself suffers from poor or a complete lack of anomeric selectivity. Nucleosides and their synthetically produced protected analogues can exist in both α- and β-anomeric forms, but only the β-anomer is usually desired for biological applications. Although the stereochemistry of the anomeric chiral centre is set in the key glycosyl donor and nucleobase coupling reaction, the inventors discovered that under certain conditions that can be used in the manufacture of decitabine, the chiral centre can epimerize (isomerise).
See, e.g., the following references:    (1) J. A. Beisler, J. Med. Chem., 1978, 21, 204.    (2) L. D. Kissinger and N. L. Stemm, J. Chromatography, 1986, 353, 309-318.    (3) a) U.S. Pat. No. 3,350,388 (1967) and DE1922702 (1969), {hacek over (S)}orm and Pískala (Ceskosl Ovenska Akademieved) and A. Pískala and F. {hacek over (S)}orm, Nucl. Acid Chem., 1978, 1, 444-449.; b) A. Pískala and F. {hacek over (S)}orm, Collect. Czech. Chem. Commun. 1964, 29, 2060.    (4) M. W. Winkley and R. K. Robins, J. Org. Chem., 1970, 35, 491.    (5) Nucleic acids in chemistry and biology, Michael Blackburn, Michael Gait, David Loakes and David Williams (eds), Cambridge, UK. The Royal Society of Chemistry, 2006, Chapter 3, pp 84-85.    (6) J. Ben-Hatter and J. Jiricny, J. Org. Chem., 1986, 51, 3211-3213.    (7) DE2012888 (1971), Vorbrüggen and Niedballa (Schering AG).    (8) U. Niedballa and H. Vorbrüggen, J. Org. Chem., 1974, 39, 3672-3674.    (9) G. Gauberta, C. Mathe', J.-L. Imbacha, S. Erikssonb, S. Vincenzettic, D. Salvatoric, A. Vitac, G. Maurya, Eur. J. Med. Chem., 2000, 35 1011-1019.    (10) U.S. Pat. No. 4,082,911 (1978), Vorbrüggen (Schering Aktiengesellschaft).    (11) CN101307084A (2008) J. R. Fan et. al.
The entire content of each of the above references is incorporated herein as reference.
Pískala and {hacek over (S)}orm[3a] teach a lengthy method for the synthesis of decitabine which involves the use of reactive N-glycosylisocyanate intermediates possessing 1-β-configuration. The synthetic process (Scheme 1) comprises reacting a peracyiglycosyl isocyanate with an S-alkylisothiurea to obtain a peracyiglycosylisothiourea, condensing the latter with an orthoester of an aliphatic acid at high temperature (135° C.) to obtain hydroxy-protected glycosyl-4-alkylmercapto-2-oxo-1,2-dihydro-1,3,5-triazine followed by deprotection with sodium methoxide (NaOMe) in methanol (MeOH) followed by decationization using an ion-exchange resin. The intermediate is then aminated with ammonia (NH3) in MeOH in a sealed vessel overnight. Although based on the isocyanate, the overall yield of decitabine is about 30%, it could be difficult to store the isocyanate and its use might provide a health risk. This isocyanate itself is produced from a chlorosugar precursor by reaction with silver cyanate.[3b] The route also suffers from other difficult to scale-up steps, including the use of the carcinogenic ICH Class I solvent benzene, and the need for a pressure vessel in the deprotection step.

Another potential process for decitabine synthesis was reported by Winkley and Robins[4] (Scheme 2). Their approach utilizes the non-catalysed coupling of a 1-chlorosugar with 2-[(trimethylsilyl)amino]-4-[(trimethylsilyl)oxy]-s-triazine (silyl 5-azacytosine) which probably proceeds via an SN2 mechanism. The yield of the desired β-anomer was very low (7% overall yield) and the process required gaseous hydrogen chloride in the synthesis of the 1-chlorosugar, long reaction times (4-5 days), the need for pressure vessels, complicated column chromatography and lengthy work-up and isolation procedures. Also, 1-halosugars (halogenoses) are not stable. There is no indication that any anomeric selectivity is obtained in this process.

Niedballa and Vorbrüggen[7,8] teach the synthesis of protected (blocked) nucleosides including decitabine that utilizes tin chloride in dichloroethane (DCE) to accelerate the coupling reaction of silyl 5-azacytosine and a protected 1-chlorosugar (Scheme 3). Even though the authors used an anomerically-enriched chlorosugar (α-anomer), an anomeric mixture of protected decitabine isomers was formed. This process suffers from difficulties in removal of tin from the API and emulsions during the aqueous work-up of the coupling mixture. Therefore, this process may not be suitable for the commercial manufacture of decitabine. Due to the sensitivity of the 5-azacytosine ring to water, any process that suffers from the formation of emulsions may potentially provide lower yields and purities of the product due to hydrolysis.
Ben-Hatter and Jiricny[6] also utilise a 1-chlorosugar in a tin chloride catalysed coupling reaction in DCE. To avoid difficulties with the hydrolysis of the sensitive 5-azacytosine ring, the authors instead used Fmoc hydroxy protection groups since these may be removed under non-aqueous, mildly basic conditions. The coupling reaction produced a 1:0.9 mixture, following silica gel chromatography, of the undesired α-anomer and the desired β-anomer of the Fmoc protected decitabine, with the latter in 21% yield based on the 1-chlorosugar (Scheme 3). A drawback of this process is that not only is the protected decitabine isolated as a mixture of anomers, but also the crude decitabine is required fractional crystallization to obtain the desired anomer.

Vorbrüggen[10] teaches a general method for the coupling of silylated heterocyclic organic bases (including cytosine, pyridines triazoles, and pyrimidines, but not 5-azacytosine) with protected 1-O-acyl, 1-O-alkyl or 1-halo-sugars (viz., ribose, deoxyribose, arabinose and glucose derivatives) in benzene, DCE or MeCN to make protected nucleosides (Scheme 4). Decitabine is not specifically described in Vorbrüggen. The coupling is promoted by trimethylsilyl (TMS) esters of esterifiable mineral acids or strong sulfonic acids, including trimethylsilyl triflate (TMSOTf), TMSOClO3 and TMSOSO2F. The use of these silyl ester catalysts in place of tin chloride is an advance in this type of chemistry, because it means that APIs may potentially be manufactured free of tin residues.

As shown above, controlling the stereochemistry of the C1 (anomeric centre) during the synthesis of 2-deoxy-ribose based nucleosides is a challenge.[5] Maury et al.[9] attempt to use a deoxygenation approach, following the direct coupling of silyl 5-azacytosine with a tetraacyl protected ribofuranose sugar, to synthesize the enantiomer of decitabine (ent-decitabine). See Scheme 5 below. Specifically, Maury et al synthesize non-2-deoxy-ribose nucleosides (i.e., synthesis of ribose based nucleosides) followed by deoxygenation of the C2′ position. In this way, the synthesis of ent-decitabine proceeds via the related nucleoside ent-azacitidine. The drawback of this approach is that the very expensive Markiewicz reagent (1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane) and tris(trimethylsilyl)silane are used in the deoxygenation part of the synthesis. Column chromatography was also used in most of the steps. The use of expensive silicon-based reagents and the extra synthetic steps required beyond those of a typical nucleoside synthesis make this approach less attractive on a manufacturing scale.

Fan et al.[11] coupled silyl 5-azacytosine and 1-O-acetyl-3,5-di-O-(2-methoxyacetyl)-2-deoxy-D-ribofuranose in toluene at 30-35° C. in the presence of a greater than stoichiometric amount of TMSOTf to provide a protected decitabine as a mixture of anomers (Scheme 6) in only 28% yield. The protected decitabine was deprotection using sodium ethoxide to give decitabine in a low 22% based on the protected decitabine, and a very low 6% overall yield.

Therefore, there is still a need for a simpler and less expensive process for producing a decitabine on a manufacture scale in a high yield and purity.